The zinc metalloproteases from Botulinum neurotoxin (BoNT) and the Lethal Toxin (LT) of B. anthracis are critical for the toxicity of their host organisms and are excellent pharmaceutical targets. Both toxins are of interest as they are components of category A biothreat agents, but the botulinum toxins are particularly significant as they are commonly used pharmaceuticals with the potential for tragic misuse. To discover active compounds for these proteases we developed a high throughput flow cytometry based protease assay to perform pilot screens of the Prestwick Library, which identified 3 lead inhibitors for Botulinum neurotoxin type A light chain protease (BoNT/A LC) and 2 lead inhibitors for the Lethal Factor protease (LF) of B. anthracis LT. We have confirmed that Ebselen is a promising inhibitor specific for BoNT/A LC with an IC50 of 5 5M. Based on our promising screening results we propose to perform extensive high throughput screening for inhibitory compounds against BoNT types A &F and LF in collaboration with the University of New Mexico Center for Molecular Discovery. Our screening assay uses high throughput flow cytometry to measure the cleavage of fluorescent fusion proteins that are attached to multiplexed microspheres. Beyond the speed at which compounds are screened, this assay is notable because it measures the activity of several proteases simultaneously across multiple full-length substrates. This offers several advantages that include reduced screening costs, homogenous endpoint activity assays, detection of inhibitors targeting known distal binding elements required for full protease activity, and immediate estimation of compound specificity via the inhibition pattern against different proteases. Lead compounds discovered in our screens will be confirmed via activity measurements in follow-up microsphere assays and two FRET protease assays, which use a simple FRET peptide or a fluorescently tagged avidin and our biotinylated fusion proteins containing full-length protease substrate and a terminal GFP tag. Our second FRET assay supports the use of full-length protease substrates, is solution based, and can be performed on fluorescence plate readers. Finally, promising compounds will be tested in cellular toxicity assays. LF toxicity assays will be performed on cells treated with LT and viability measured through luminescent readout of ATP production. We will test compounds targeting BoNT/A &F LC by administering BoNT/A &F LC to mammalian cells that intracellularly express fusion protein bearing CFP and YFP separated by a SNAP-25 or VAMP-2 substrate. Compound activity will be monitored via flow cytometry as a reduction of the loss of CFP to YFP FRET as compared to untreated cells. As a final assay, BoNT inhibitors that pass through this process will be compared in dose response assays against other commercially available BoNT LCs (B, C, D, &E) that are predicted to be active on all of our substrates. Due to the significant homology of all the BoNTs, we expect significant cross reactivity to these other proteases. The combination of our novel screening approach with our confirmation assays will enable the rapid mulitplexed discovery of active compounds for BoNT/A &F LC and LF using a simple set of screens that implements three proteases simultaneously to dramatically reduce cost and labor. Furthermore, our approach has the novel ability to use full-length substrates that will enable the discovery of inhibitor compounds that target protease interactions with substrate binding sites distal to the active site. Thus, this screening proposal offers an inexpensive approach to rapidly discover new inhibitors including novel inhibitor types that would be invisible by other methods. These molecules will be valuable to pharmaceutical development efforts, biological projects focused on protease kinetics at surfaces, and potentially as probes for the SNARE protein complex formation in the neuronal exocytosis pathway. PUBLIC HEALTH RELEVANCE: This proposal will discover new and important molecules that can serve as specific activity probes and inhibitors for the important toxin proteases from Botulinum neurotoxin and Bacillus anthracis. In this way, this proposal will discover important new compounds that can be the basis of new pharmaceuticals to treat Botulinum neurotoxin poisoning or late stage Anthrax infections.